The primary objective of this application is to determine if functional CD4+CD25+ regulatory T cells exist in CD40-/- BALB/c mice. We have found that CD40-/- BALB/c mice develop a significantly reduced population of T cells with the CD4+CD25+ cell surface phenotype compared to wildtype BALB/c mice. Conventionally raised, adult CD40-/- BALB/c have less than 75% of the number of CD4+CD25+ T cells as found in wildtype BALB/c mice. We have evaluated the CD4 and CD8 T-cell response that develops in CD40 -/- BALB/c mice following immunization with Listeria monocytogenes and have compared this response with the Listeriaderived peptide-specific response that develops in similarly immunized wildtype BALB/c mice. The Listeriaspecific response found in CD4 and CD8 T-cell populations is significantly elevated in CD40-/- BALB/c mice compared to the response that develops in wildtype BALB/c mice. This response differential is evident at the primary response and is especially notable during the secondary response. We have also found that in response to high bacterial numbers at the time of secondary challenge CD40-/- BALB/c mice die even though the challenge dose of L. monocytogenes has been cleared, suggesting an unregulated response to secondary challenge and a detrimental production of proinflammatory cytokines. These observations imply that the low numbers of CD4+CD25+ T cells found in CD40-/- BALB/c mice result in the development of a less or completely unregulated immune response following challenge with L. monocytogenes. This observation is in keeping with the currently held view of CD4+CD25+ T cells as a resident immunoregulatory cell subset that influences the antigen-dependent response of both CD4 and CD8 T-cell populations. In this R-21 application we propose to analyze in greater detail the features and the consequence of the reduction of this regulatory T cell subset as it exists in CD40-/- BALB/c mice. Specifically we will determine if the residual CD4+CD25+ T cell population retains immunoregulatory function and will also determine if CD4+CD25+ T cells derived from wildtype BALB/c mice can control the response of CD4 and CD8 T cell populations that have developed within the CD40-/- environment. Improved means/methods are needed to study the many features of CD4+CD25+ regulatory T cells. The CD40-/- BALB/c mouse strain may prove to be a preferred model for expanded studies on the development and function of this immunoregulatory T-cell subset.